A population of naive-like CD4+ T cells stably polarized to the TH 1 lineage.

T-bet is the lineage-specifying transcription factor for CD4+ TH 1 cells. T-bet has also been found in other CD4+ T cell subsets, including TH 17 cells and Treg, where it modulates their functional characteristics. However, we lack information on when and where T-bet is expressed during T cell differentiation and how this impacts T cell differentiation and function. To address this, we traced the ontogeny of T-bet-expressing cells using a fluorescent fate-mapping mouse line. We demonstrate that T-bet is expressed in a subset of CD4+ T cells that have naïve cell surface markers and transcriptional profile and that this novel cell population is phenotypically and functionally distinct from previously described populations of naïve and memory CD4+ T cells. Naïve-like T-bet-experienced cells are polarized to the TH 1 lineage, predisposed to produce IFN-γ upon cell activation, and resist repolarization to other lineages in vitro and in vivo. These results demonstrate that lineage-specifying factors can polarize T cells in the absence of canonical markers of T cell activation and that this has an impact on the subsequent T-helper response.

Pluripotent Stem Cell-Derived Hepatocytes Inhibit T Cell Proliferation In Vitro through Tryptophan Starvation.

Regenerative medicine aims to replace damaged tissues by stimulating endogenous tissue repair or by transplanting autologous or allogeneic cells. Due to their capacity to produce unlimited numbers of cells of a given cell type, pluripotent stem cells, whether of embryonic origin or induced via the reprogramming of somatic cells, are of considerable therapeutic interest in the regenerative medicine field. However, regardless of the cell type, host immune responses present a barrier to success. The aim of this study was to investigate in vitro the immunological properties of human pluripotent stem cell (PSC)-derived hepatocyte-like cells (HLCs). These cells expressed MHC class I molecules while they lacked MHC class II and co-stimulatory molecules, such as CD80 and CD86. Following stimulation with IFN-γ, HLCs upregulated CD40, PD-L1 and MHC class I molecules. When co-cultured with allogeneic T cells, HLCs did not induce T cell proliferation; furthermore, when T cells were stimulated via αCD3/CD28 beads, HLCs inhibited their proliferation via IDO1 and tryptophan deprivation. These results demonstrate that PSC-derived HLCs possess immunoregulatory functions, at least in vitro.

Supplementation with a prebiotic (polydextrose) in obese mouse pregnancy improves maternal glucose homeostasis and protects against offspring obesity.

OBJECTIVES: We hypothesised that maternal diet-induced-obesity has adverse consequences for offspring energy expenditure and susceptibility to obesity in adulthood, and that the prebiotic polydextrose (PDX) would prevent the consequences of programming by maternal obesity. METHODS: Female mice were fed a control (Con) or obesogenic diet (Ob) for 6 weeks prior to mating and throughout pregnancy and lactation. Half the obese dams were supplemented with 5% PDX (ObPDX) in drinking water throughout pregnancy and lactation. Offspring were weaned onto standard chow. At 3 and 6 months, offspring energy intake (EI) and energy expenditure (EE by indirect calorimetry) were measured, and a glucose-tolerance test performed. Offspring of control (OffCon), obese (OffOb) and PDX supplemented (OffObP) dams were subsequently challenged for 3 weeks with Ob, and energy balanced reassessed. Potential modifiers of offspring energy balance including gut microbiota and biomarkers of mitochondrial activity were also evaluated. RESULTS: Six-month-old male OffOb demonstrated increased bodyweight (BW, P < 0.001) and white adipose tissue mass (P < 0.05), decreased brown adipose tissue mass (BAT, P < 0.01), lower night-time EE (P < 0.001) versus OffCon, which were prevented in OffObP. Both male and female OffOb showed abnormal glucose-tolerance test (peak [Glucose] P < 0.001; AUC, P < 0.05) which was prevented by PDX. The Ob challenge resulted in greater BW gain in both male and female OffOb versus OffCon (P < 0.05), also associated with increased EI (P < 0.05) and reduced EE in females (P < 0.01). OffObP were protected from accelerated BW gain on the OB diet compared with controls, associated with increased night-time EE in both male (P < 0.05) and female OffObP (P < 0.001). PDX also prevented an increase in skeletal muscle mtDNA copy number in OffOb versus OffCon (P < 0.01) and increased the percentage of Bacteroides cells in faecal samples from male OffObP relative to controls. CONCLUSIONS: Maternal obesity adversely influences adult offspring energy balance and propensity for obesity, which is ameliorated by maternal PDX treatment with associated changes in gut microbiota composition and skeletal muscle mitochondrial function.

Interleukin-22 orchestrates a pathological endoplasmic reticulum stress response transcriptional programme in colonic epithelial cells.

OBJECTIVE: The functional role of interleukin-22 (IL22) in chronic inflammation is controversial, and mechanistic insights into how it regulates target tissue are lacking. In this study, we evaluated the functional role of IL22 in chronic colitis and probed mechanisms of IL22-mediated regulation of colonic epithelial cells. DESIGN: To investigate the functional role of IL22 in chronic colitis and how it regulates colonic epithelial cells, we employed a three-dimentional mini-gut epithelial organoid system, in vivo disease models and transcriptomic datasets in human IBD. RESULTS: As well as inducing transcriptional modules implicated in antimicrobial responses, IL22 also coordinated an endoplasmic reticulum (ER) stress response transcriptional programme in colonic epithelial cells. In the colon of patients with active colonic Crohn's disease (CD), there was enrichment of IL22-responsive transcriptional modules and ER stress response modules. Strikingly, in an IL22-dependent model of chronic colitis, targeting IL22 alleviated colonic epithelial ER stress and attenuated colitis. Pharmacological modulation of the ER stress response similarly impacted the severity of colitis. In patients with colonic CD, antibody blockade of IL12p40, which simultaneously blocks IL12 and IL23, the key upstream regulator of IL22 production, alleviated the colonic epithelial ER stress response. CONCLUSIONS: Our data challenge perceptions of IL22 as a predominantly beneficial cytokine in IBD and provide novel insights into the molecular mechanisms of IL22-mediated pathogenicity in chronic colitis. Targeting IL22-regulated pathways and alleviating colonic epithelial ER stress may represent promising therapeutic strategies in patients with colitis. TRIAL REGISTRATION NUMBER: NCT02749630.

Adipose tissue inflammation in obesity: a metabolic or immune response?

Adipose tissue is not only a reservoir for energy, but also an immune organ. In the context of obesity, the development of insulin resistance is now recognised to be initiated by inflammation of the adipose tissue. However, the primary events triggering this inflammation are still unclear, as a complex combination of endocrine and immune factors act to regulate this adipose tissue microenvironment. Below we discuss the different factors involved and how they affect the biology of the adipose tissue in obesity.

Fermentable carbohydrate stimulates FFAR2-dependent colonic PYY cell expansion to increase satiety.

OBJECTIVE: Dietary supplementation with fermentable carbohydrate protects against body weight gain. Fermentation by the resident gut microbiota produces short-chain fatty acids, which act at free fatty acid receptor 2 (FFAR2). Our aim was to test the hypothesis that FFAR2 is important in regulating the beneficial effects of fermentable carbohydrate on body weight and to understand the role of gut hormones PYY and GLP-1. METHODS: Wild-type or Ffar2-/- mice were fed an inulin supplemented or control diet. Mice were metabolically characterized and gut hormone concentrations, enteroendocrine cell density measurements were carried out. Intestinal organoids and colonic cultures were utilized to substantiate the in vivo findings. RESULTS: We provide new mechanistic insight into how fermentable carbohydrate regulates metabolism. Using mice that lack FFAR2, we demonstrate that the fermentable carbohydrate inulin acts via this receptor to drive an 87% increase in the density of cells that produce the appetite-suppressing hormone peptide YY (PYY), reduce food intake, and prevent diet-induced obesity. CONCLUSION: Our results demonstrate that FFAR2 is predominantly involved in regulating the effects of fermentable carbohydrate on metabolism and does so, in part, by enhancing PYY cell density and release. This highlights the potential for targeting enteroendocrine cell differentiation to treat obesity.

Developing in vitro expanded CD45RA+ regulatory T cells as an adoptive cell therapy for Crohn's disease.

BACKGROUND AND AIM: Thymus-derived regulatory T cells (Tregs) mediate dominant peripheral tolerance and treat experimental colitis. Tregs can be expanded from patient blood and were safely used in recent phase 1 studies in graft versus host disease and type 1 diabetes. Treg cell therapy is also conceptually attractive for Crohn's disease (CD). However, barriers exist to this approach. The stability of Tregs expanded from Crohn's blood is unknown. The potential for adoptively transferred Tregs to express interleukin-17 and exacerbate Crohn's lesions is of concern. Mucosal T cells are resistant to Treg-mediated suppression in active CD. The capacity for expanded Tregs to home to gut and lymphoid tissue is unknown. METHODS: To define the optimum population for Treg cell therapy in CD, CD4(+)CD25(+)CD127(lo)CD45RA(+) and CD4(+)CD25(+)CD127(lo)CD45RA(-) Treg subsets were isolated from patients' blood and expanded in vitro using a workflow that can be readily transferred to a good manufacturing practice background. RESULTS: Tregs can be expanded from the blood of patients with CD to potential target dose within 22-24 days. Expanded CD45RA(+) Tregs have an epigenetically stable FOXP3 locus and do not convert to a Th17 phenotype in vitro, in contrast to CD45RA(-) Tregs. CD45RA(+) Tregs highly express α4ß7 integrin, CD62L and CC motif receptor 7 (CCR7). CD45RA(+) Tregs also home to human small bowel in a C.B-17 severe combined immune deficiency (SCID) xenotransplant model. Importantly, in vitro expansion enhances the suppressive ability of CD45RA(+) Tregs. These cells also suppress activation of lamina propria and mesenteric lymph node lymphocytes isolated from inflamed Crohn's mucosa. CONCLUSIONS: CD4(+)CD25(+)CD127(lo)CD45RA(+) Tregs may be the most appropriate population from which to expand Tregs for autologous Treg therapy for CD, paving the way for future clinical trials.

Cutting Edge: Retinoic Acid Signaling in B Cells Is Essential for Oral Immunization and Microflora Composition.

Retinoic acid (RA) is a critical regulator of the intestinal adaptive immune response. However, the intrinsic impact of RA on B cell differentiation in the regulation of gut humoral immunity in vivo has never been directly shown. To address this issue, we have been able to generate a mouse model where B cells specifically express a dominant-negative receptor α for RA. In this study, we show that the silencing of RA signaling in B cells reduces the numbers of IgA(+) Ab-secreting cells both in vitro and in vivo, suggesting that RA has a direct effect on IgA plasma cell differentiation. Moreover, the lack of RA signaling in B cells abrogates Ag-specific IgA responses after oral immunization and affects the microbiota composition. In conclusion, these results suggest that RA signaling in B cells through the RA receptor α is important to generate an effective gut humoral response and to maintain a normal microbiota composition.

Interleukin 6 Increases Production of Cytokines by Colonic Innate Lymphoid Cells in Mice and Patients With Chronic Intestinal Inflammation.

BACKGROUND & AIMS: Innate lymphoid cells (ILCs) are a heterogeneous group of mucosal inflammatory cells that participate in chronic intestinal inflammation. We investigated the role of interleukin 6 (IL6) in inducing activation of ILCs in mice and in human beings with chronic intestinal inflammation. METHODS: ILCs were isolated from colons of Tbx21(-/-) × Rag2(-/-) mice (TRUC), which develop colitis; patients with inflammatory bowel disease (IBD); and patients without colon inflammation (controls). ILCs were characterized by flow cytometry; cytokine production was measured by enzyme-linked immunosorbent assay and cytokine bead arrays. Mice were given intraperitoneal injections of depleting (CD4, CD90), neutralizing (IL6), or control antibodies. Isolated colon tissues were analyzed by histology, explant organ culture, and cell culture. Bacterial DNA was extracted from mouse fecal samples to assess the intestinal microbiota. RESULTS: IL17A- and IL22-producing, natural cytotoxicity receptor-negative, ILC3 were the major subset of ILCs detected in colons of TRUC mice. Combinations of IL23 and IL1α induced production of cytokines by these cells, which increased further after administration of IL6. Antibodies against IL6 reduced colitis in TRUC mice without significantly affecting the structure of their intestinal microbiota. Addition of IL6 increased production of IL17A, IL22, and interferon-γ by human intestinal CD3-negative, IL7-receptor-positive cells, in a dose-dependent manner. CONCLUSIONS: IL6 contributes to activation of colonic natural cytotoxicity receptor-negative, CD4-negative, ILC3s in mice with chronic intestinal inflammation (TRUC mice) by increasing IL23- and IL1α-induced production of IL17A and IL22. This pathway might be targeted to treat patients with IBD because IL6, which is highly produced in colonic tissue by some IBD patients, also increased the production of IL17A, IL22, and interferon-γ by cultured human colon CD3-negative, IL7-receptor-positive cells.

Transitional-2 B cells acquire regulatory function during tolerance induction and contribute to allograft survival.

In humans, tolerance to renal transplants has been associated with alterations in B-cell gene transcription and maintenance of the numbers of circulating transitional B cells. Here, we use a mouse model of transplantation tolerance to investigate the contribution of B cells to allograft survival. We demonstrate that transfer of B cells from mice rendered tolerant to MHC class I mismatched skin grafts can prolong graft survival in a dose-dependent and antigen-specific manner to a degree similar to that afforded by graft-specific regulatory T (Treg) cells. Tolerance in this model was associated with an increase in transitional-2 (T2) B cells. Only T2 B cells from tolerized mice, not naïve T2 nor alloantigen experienced T2, were capable of prolonging skin allograft survival, and suppressing T-cell activation. Tolerized T2 B cells expressed lower levels of CD86, increased TIM-1, and demonstrated a preferential survival in vivo. Furthermore, we demonstrate a synergistic effect between tolerized B cells and graft-specific Treg cells. IL-10 production by T2 B cells did not contribute to tolerance, as shown by transfer of B cells from IL-10(-/-) mice. These results suggest that T2 B cells in tolerant patients may include a population of regulatory B cells that directly inhibit graft rejection.

The immune cell transcription factor T-bet: A novel metabolic regulator.

Obesity-associated insulin resistance is accompanied by an alteration in the Th1/Th2 balance in adipose tissue. T-bet (Tbx21) is an immune cell transcription factor originally described as the master regulator of Th1 cell development, although is now recognized to have a role in both the adaptive and innate immune systems. T-bet also directs T-cell homing to pro-inflammatory sites by the regulation of CXCR3 expression. T-bet(-/-) mice have increased visceral adiposity but are more insulin-sensitive, exhibiting reduced immune cell content and cytokine secretion specifically in the visceral fat depot, perhaps due to altered T-cell trafficking. Studies of T-bet deficiency on Rag2-- and IFN-γ-deficient backgrounds indicate the importance of CD4(+) T cells and IFN-γ in this model. This favorable metabolic phenotype, uncoupling adiposity from insulin resistance, is present in young lean mice yet persists with age and increasing obesity. We suggest a novel role for T-bet in metabolic regulation.

Improved insulin sensitivity despite increased visceral adiposity in mice deficient for the immune cell transcription factor T-bet.

Low-grade inflammation in fat is associated with insulin resistance, although the mechanisms are unclear. We report that mice deficient in the immune cell transcription factor T-bet have lower energy expenditure and increased visceral fat compared with wild-type mice, yet paradoxically are more insulin sensitive. This striking phenotype, present in young T-bet(-/-) mice, persisted with high-fat diet and increasing host age and was associated with altered immune cell numbers and cytokine secretion specifically in visceral adipose tissue. However, the favorable metabolic phenotype observed in T-bet-deficient hosts was lost in T-bet(-/-) mice also lacking adaptive immunity (T-bet(-/-)xRag2(-/-)), demonstrating that T-bet expression in the adaptive rather than the innate immune system impacts host glucose homeostasis. Indeed, adoptive transfer of T-bet-deficient, but not wild-type, CD4(+) T cells to Rag2(-/-) mice improved insulin sensitivity. Our results reveal a role for T-bet in metabolic physiology and obesity-associated insulin resistance.

LXR activation by GW3965 alters fat tissue distribution and adipose tissue inflammation in ob/ob female mice.

To investigate the role of liver X receptor (LXR) in adipose tissue metabolism during obesity, ob/ob mice were treated for 5 weeks with the synthetic LXR agonist GW3965. MRI analysis revealed that pharmacological activation of LXR modified fat distribution by decreasing visceral (VS) fat and inversely increasing subcutaneous (SC) fat storage without affecting whole body fat content. This was concordant with opposite regulation by GW3965 of the lipolytic markers hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) in the two fat depots; moreover, the expression of genes involved in lipogenesis was significantly induced in SC fat. Lipidomic analysis suggested that changes in lipid composition in response to GW3965 also varied between VS and SC fat. In both depots, the observed alteration in lipid composition indicated an overall change toward less lipotoxic lipids. Flow cytometry analysis showed decreased immune cell infiltration in adipose tissue of ob/ob mice in response to GW3965 treatment, which in VS fat mainly affected the macrophage population and in SC fat the lymphocyte population. In line with this, the expression and secretion of proinflammatory markers was decreased in both fat deposits with GW3965 treatment.

The transcription factor T-bet regulates intestinal inflammation mediated by interleukin-7 receptor+ innate lymphoid cells.

Mice lacking the transcription factor T-bet in the innate immune system develop microbiota-dependent colitis. Here, we show that interleukin-17A (IL-17A)-producing IL-7Rα(+) innate lymphoid cells (ILCs) were potent promoters of disease in Tbx21(-/-)Rag2(-/-) ulcerative colitis (TRUC) mice. TNF-α produced by CD103(-)CD11b(+) dendritic cells synergized with IL-23 to drive IL-17A production by ILCs, demonstrating a previously unrecognized layer of cellular crosstalk between dendritic cells and ILCs. We have identified Helicobacter typhlonius as a key disease trigger driving excess TNF-α production and promoting colitis in TRUC mice. Crucially, T-bet also suppressed the expression of IL-7R, a key molecule involved in controlling intestinal ILC homeostasis. The importance of IL-7R signaling in TRUC disease was highlighted by the dramatic reduction in intestinal ILCs and attenuated colitis following IL-7R blockade. Taken together, these data demonstrate the mechanism by which T-bet regulates the complex interplay between mucosal dendritic cells, ILCs, and the intestinal microbiota.

Detection of extracellular glucose by GLUT2 contributes to hypothalamic control of food intake.

The sugar transporter GLUT2, present in several tissues of the gut-brain axis, has been reported to be involved in the control of food intake. GLUT2 is a sugar transporter sustaining energy production in the cell, but it can also function as a receptor for extracellular glucose. A glucose-signaling pathway is indeed triggered, independently of glucose metabolism, through its large cytoplasmic loop domain. However, the contribution of the receptor function over the transporter function of GLUT2 in the control of food intake remains to be determined. Thus, we generated transgenic mice that express a GLUT2-loop domain, blocking the detection of glucose but leaving GLUT2-dependent glucose transport unaffected. Inhibiting GLUT2-mediated glucose detection augmented daily food intake by a mechanism that increased the meal size but not the number of meals. Peripheral hormones (ghrelin, insulin, leptin) were unaffected, leading to a focus on central aspects of feeding behavior. We found defects in c-Fos activation by glucose in the arcuate nucleus and changes in the amounts of TRH and orexin neuropeptide mRNA, which are relevant to poorly controlled meal size. Our data provide evidence that glucose detection by GLUT2 contributes to the control of food intake by the hypothalamus. The sugar transporter receptor, i.e., "transceptor" GLUT2, may constitute a drug target to treat eating disorders and associated metabolic diseases, particularly by modulating its receptor function without affecting vital sugar provision by its transporter function.

Characterisation of glutamine fructose-6-phosphate amidotransferase (EC 2.6.1.16) and N-acetylglucosamine metabolism in Bifidobacterium.

Bifidobacterium bifidum, in contrast to other bifidobacterial species, is auxotrophic for N-acetylglucosamine. Growth experiments revealed assimilation of radiolabelled N-acetylglucosamine in bacterial cell walls and in acetate, an end-product of central metabolism via the bifidobacterial D: -fructose-6-phosphate shunt. While supplementation with fructose led to reduced N-acetylglucosamine assimilation via the D: -fructose-6-phosphate shunt, no significant difference was observed in levels of radiolabelled N-acetylglucosamine incorporated into cell walls. Considering the central role played by glutamine fructose-6-phosphate transaminase (GlmS) in linking the biosynthetic pathway for N-acetylglucosamine to hexose metabolism, the GlmS of Bifidobacterium was characterized. The genes encoding the putative GlmS of B. longum DSM20219 and B. bifidum DSM20082 were cloned and sequenced. Bioinformatic analyses of the predicted proteins revealed 43% amino acid identity with the Escherichia coli GlmS, with conservation of key amino acids in the catalytic domain. The B. longum GlmS was over-produced as a histidine-tagged fusion protein. The purified C-terminal His-tagged GlmS possessed glutamine fructose-6-phosphate amidotransferase activity as demonstrated by synthesis of glucosamine-6-phosphate from fructose-6-phosphate and glutamine. It also possesses an independent glutaminase activity, converting glutamine to glutamate in the absence of fructose-6-phosphate. This is of interest considering the apparently reduced coding potential in bifidobacteria for enzymes associated with glutamine metabolism.

Insulin internalizes GLUT2 in the enterocytes of healthy but not insulin-resistant mice.

OBJECTIVES: A physiological adaptation to a sugar-rich meal is achieved by increased sugar uptake to match dietary load, resulting from a rapid transient translocation of the fructose/glucose GLUT2 transporter to the brush border membrane (BBM) of enterocytes. The aim of this study was to define the contributors and physiological mechanisms controlling intestinal sugar absorption, focusing on the action of insulin and the contribution of GLUT2-mediated transport. RESEARCH DESIGN AND METHODS: The studies were performed in the human enterocytic colon carcinoma TC7 subclone (Caco-2/TC7) cells and in vivo during hyperinsulinemic-euglycemic clamp experiments in conscious mice. Chronic high-fructose or high-fat diets were used to induce glucose intolerance and insulin resistance in mice. RESULTS AND CONCLUSIONS: In Caco-2/TC7 cells, insulin action diminished the transepithelial transfer of sugar and reduced BBM and basolateral membrane (BLM) GLUT2 levels, demonstrating that insulin can target sugar absorption by controlling the membrane localization of GLUT2 in enterocytes. Similarly, in hyperinsulinemic-euglycemic clamp experiments in sensitive mice, insulin abolished GLUT2 (i.e., the cytochalasin B-sensitive component of fructose absorption), decreased BBM GLUT2, and concomitantly increased intracellular GLUT2. Acute insulin treatment before sugar intake prevented the insertion of GLUT2 into the BBM. Insulin resistance in mice provoked a loss of GLUT2 trafficking, and GLUT2 levels remained permanently high in the BBM and low in the BLM. We propose that, in addition to its peripheral effects, insulin inhibits intestinal sugar absorption to prevent excessive blood glucose excursion after a sugar meal. This protective mechanism is lost in the insulin-resistant state induced by high-fat or high-fructose feeding.

Loss of sugar detection by GLUT2 affects glucose homeostasis in mice.

BACKGROUND: Mammals must sense the amount of sugar available to them and respond appropriately. For many years attention has focused on intracellular glucose sensing derived from glucose metabolism. Here, we studied the detection of extracellular glucose concentrations in vivo by invalidating the transduction pathway downstream from the transporter-detector GLUT2 and measured the physiological impact of this pathway. METHODOLOGY/PRINCIPAL FINDINGS: We produced mice that ubiquitously express the largest cytoplasmic loop of GLUT2, blocking glucose-mediated gene expression in vitro without affecting glucose metabolism. Impairment of GLUT2-mediated sugar detection transiently protected transgenic mice against starvation and streptozotocin-induced diabetes, suggesting that both low- and high-glucose concentrations were not detected. Transgenic mice favored lipid oxidation, and oral glucose was slowly cleared from blood due to low insulin production, despite massive urinary glucose excretion. Kidney adaptation was characterized by a lower rate of glucose reabsorption, whereas pancreatic adaptation was associated with a larger number of small islets. CONCLUSIONS/SIGNIFICANCE: Molecular invalidation of sugar sensing in GLUT2-loop transgenic mice changed multiple aspects of glucose homeostasis, highlighting by a top-down approach, the role of membrane glucose receptors as potential therapeutic targets.

Sugar sensing by enterocytes combines polarity, membrane bound detectors and sugar metabolism.

Sugar consumption and subsequent sugar metabolism are known to regulate the expression of genes involved in intestinal sugar absorption and delivery. Here we investigate the hypothesis that sugar-sensing detectors in membranes facing the intestinal lumen or the bloodstream can also modulate intestinal sugar absorption. We used wild-type and GLUT2-null mice, to show that dietary sugars stimulate the expression of sucrase-isomaltase (SI) and L-pyruvate kinase (L-PK) by GLUT2-dependent mechanisms, whereas the expression of GLUT5 and SGLT1, did not rely on the presence of GLUT2. By providing sugar metabolites, sugar transporters, including GLUT2, fuelled a sensing pathway. In Caco2/TC7 enterocytes, we could disconnect the sensing triggered by detector from that produced by metabolism, and found that GLUT2 generated a metabolism-independent pathway to stimulate the expression of SI and L-PK. In cultured enterocytes, both apical and basolateral fructose could increase the expression of GLUT5, conversely, basolateral sugar administration could stimulate the expression of GLUT2. Finally, we located the sweet-taste receptors T1R3 and T1R2 in plasma membranes, and we measured their cognate G alpha Gustducin mRNA levels. Furthermore, we showed that a T1R3 inhibitor altered the fructose-induced expression of SGLT1, GLUT5, and L-PK. Intestinal gene expression is thus controlled by a combination of at least three sugar-signaling pathways triggered by sugar metabolites and membrane sugar receptors that, according to membrane location, determine sugar-sensing polarity. This provides a rationale for how intestine adapts sugar delivery to blood and dietary sugar provision.